The goals of this project are to develop a DNA vaccine formulationthat effectivelystimulates all arms of the immune response mechanismand is applicableto a human HIV-1 DNA vaccine. The novel focus of this research is to target the antigen as a chimera containing sequences of the lysosome membrane protein (LAMP) that traffick the chimera to the major histocompatability class II (MHC II) compartment for antigen processing and presention. This approach is designed to increase antigen-specific CD4+ T-helper (Th) lymphocyte responses which are critical for optimal CD8+and humoral immune responses. Our research with the LAMP/Gag chimera vaccine has shown that targeting of the antigen to the MHC II compartment results in a marked increase in all arms of the immune responses of mice, antibody, CD4+helper T cells, and CD8+cytotoxic T cells, with increases in CD4 antibody responses 10- to 100-fold greater than those resulting from DNA encoding wild type antigen. Our proposed research has the following specific aims: (1) Analysis of primate immune responses to LAMP-targeted HIV-I Gag is being conducted under the auspices of the NIAID to determine if the enhanced immune responses of mice to DNA encoding an HIV-1 LAMP/Gag chimera is applicable to a non-human primate. We will synthesize the human LAMP/Gag chimera, validate the correct expression and trafficking of the antigen, and assay relevant immune responses of the immunized monkeys. The synthesis and analysis of MHC II targeting of DNA vaccines encoding Env, Rev, Tat, and Nef as other candidates for possible human clinical studies will also be initiated. (2) Dendritic cell specific proteins, DC-LAMP and C-type lectin endocytic receptors, that also traffick to the MHC II compartment and demonstrate properties as Gag chimeras that differ from the multicellular LAMP targeting system, are being analyzed for possibly unique immune response mechanisms that may prove of value to a HIV-1 DNA vaccine formulation. (3) Evaluation of adeno-associated virus vector (AAV), one of the most promising gene delivery vehicles, is being conducted with our DNA vaccine formulations. Initial promising results of prolonged immunogenicity and enhanced antibody response to a DNA prime, AAV boost regimen, encourage continued investigation. PERFORMANCESITE(S) (organization,city, state) The Johns Hopkins UniversitySchoolof Medicine,Baltimore,Maryland Johns HopkinsSingaporeBiomedicalCentre, Singapore KEY PERSONNEL. See instructions. Use continuationpages as needed to providethe requiredinformationinthe format shownbelow. Startwith Principal Investigator.List all other key personnelin alphabeticalorder, last namefirst. Name Organization Roleon Project August, J. Thomas,MD Johns HopkinsUniv.Schoolof Medicine Principal Investigator Marques,Ernesto,MD/PhD Johns HopkinsUniv.Schoolof Medicine ResearchAssociate Chikhlikar,Priya, PhD Johns HopkinsUniv.Schoolof Medicine PostdoctoralFellow Leao, Ihid,MD Johns HopkinsUniv.Schoolof Medicine PostdoctoralFellow Sim Li Chen, Del, PhD Johns HopkinsSingaporeBiomed.Centre PostdoctoralFellow Arruda-Hinds,Luciana Johns HopkinsUniv.Schoolof Medicine Predoctoralstudent Maciel,MiltonJunior, PhD Johns HopkinsSingaporeBiomed.Centre PostdoctoralFellow Disclosure Permission Statement. Applicableto SBIR/STTROnly. See instructions.[] Yes [] No + PHS 398 (Rev. 05/01) Page 2 Form Page 2 +Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b. h Principal Investigator/ProgramDirector(Last,first, middle): August, J. Thomas The nameof the principalinvestigator/programdirector must beprovided at the top of each printedpage andeach continuationpage. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page ......................................................................................................................................................... 1 Description,